Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Clinical Chemistry Branch, Atlanta, Georgia 30341, USA.
Clin Chem. 2012 Apr;58(4):768-76. doi: 10.1373/clinchem.2011.177063. Epub 2012 Jan 24.
The CDC's Lipid Standardization Program established the chromotropic acid (CA) reference measurement procedure (RMP) as the accuracy base for standardization and metrological traceability for triglyceride testing. The CA RMP has several disadvantages, including lack of ruggedness. It uses obsolete instrumentation and hazardous reagents. To overcome these problems the CDC developed an isotope dilution GC-MS (ID-GC-MS) RMP for total glycerides in serum.
We diluted serum samples with Tris-HCl buffer solution and spiked 200-μL aliquots with [(13)C(3)]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b).
The calibration response was linear for the range of calibrator concentrations examined (0-1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123-0.7225) and 0.3122 (95% CI, 0.3096-0.3140), respectively. The limit of detection was 14.8 μmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%.
This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.
疾病预防控制中心的脂质标准化计划将变色酸（CA）参考测量程序（RMP）确立为甘油三酯检测的标准化和计量溯源的准确度基础。CA RMP 存在一些缺点，包括缺乏坚固性。它使用过时的仪器和危险试剂。为了克服这些问题，疾病预防控制中心为血清中的总甘油三酯开发了一种同位素稀释气相色谱-质谱（ID-GC-MS）RMP。
我们用 Tris-HCl 缓冲溶液稀释血清样本，并在 200μL 等分试样中加入 [(13)C(3)]-甘油。这些样品在碱性条件下孵育和水解。将样品干燥，用乙酸酐和吡啶衍生化，用乙酸乙酯提取，然后通过 ID-GC-MS 进行分析。通过分析校准剂溶液、10 个血清池和标准参考物质（SRM 1951b）来评估线性度、不精密度和准确度。
校准响应在检查的校准剂浓度范围内呈线性（0-1.24mmol/L），斜率和截距分别为 0.717（95%置信区间，0.7123-0.7225）和 0.3122（95%置信区间，0.3096-0.3140）。检测限为 14.8μmol/L。样本集（血清池和 SRM）的平均%CV 为 1.2%。SRM 1951b 与 NIST 同位素稀释 MS 值的平均%偏差为 0.7%。
该 ID-GC-MS RMP 具有特异性和坚固性，可准确定量脂质标准化计划中使用的血清池中的总甘油三酯，并与 NIST 总甘油三酯测量的主要 RMP 具有足够的一致性。
